Analyses statistiques

Les résultats ont été présentés comme moyenne ± SE et les données ont été analysées à l’aide de test T.student. La valeur P < 0.05 est considérée comme significative.

79

80

Article1.

Ovarian ascites-derived hospicells promote up regulation of the ATP-binding

cassette genes by Insulin-like Growth Factor-I via JAK-STAT3, MEK

signaling pathway in ovarian cancer cell line

Benabbou N., Mirshahi P., Azzazene D., Cadillon M., Soria J., Therwat A., Mirshahi M.

Dans ce travail, nous avons étudié le rôle de IGF-I secrété par le microenvironnement sur la

chimiorésistance induite par l’interaction de cellules du cancer de l’ovaire et les hospicells isolées

à partir des ascites.

Nous avons montré que les hospicells et les cellules cancéreuses de l’ovaire expriment et secrètent IGF-I.

Nous avons également montré que le facteur de croissance IGF-I est impliqué dans la régulation des gènes ABC (MDR1, MRP1, MRP2 et MRP3) très actifs dans la chimiorésistance, via les voies de signalisations STAT3, PI3K , JAK2 et MEK dans les cellules du cancer de

l’ovaire.

Une co-culture entre les cellules du cancer de l’ovaire et les hospicells, induit une chimiorésistance des cellules tumorales contre la carbopltine et taxol. Cette chimiorésistance et amplifiée par IGF-I et diminuée en inhibant son récepteur IGF-IR. A l’inverse, lorsqu’il n’y a pas de contact hospicells-cellules tumorales, et que ces dernières ne sont qu’en présence de surnageant des hospicells, les cellules cancéreuses ne résistent que légèrement aux agents de la chimiothérapie, et ce, avec ou en absence de IGF-I.

Ces résultats suggèrent que la chimiorésistance des cellules tumorales de l’ovaire est plus

importante lorsqu’il y a une synergie des effets induits par un niveau suffisant de IGF-I, secrété par le microenvironnement, et par l’interconnexion hospicells-cellules tumorales.

81

Ovarian ascites-derived hospicells promote up regulation of the ATP-binding

cassette genes by Insulin-like Growth Factor-I via JAK-STAT3, MEK

signaling pathway in ovarian cancer cell line

Nadia Benabbou, Pezhman Mirshahi, Dalel Azazzene, Mélodie Cadillon, Jeannette Soria, Amu Therwat, Massoud Mirshahi*

National Institut for Medical Research (INSERM), Cordeliers Research Center (UMRS 872), University of Pierre and Marie Curie, and University of Paris Descartes.

Running Title: Hospicells promote up regulation of ABC genes by IGF-1in ovarian cancer

Key words: IGF-1, JAK-STAT3, MEK, PI-3Kinase Signaling pathway, ATP Binding Cassette, Hospicells, chemoresistance, ovarian cancer,

*Corresponding Author:

M. Mirshahi, M. D., Ph. D. INSERM UMRS 872, Université Pierre et Marie Curie, Paris-VI,

Université Paris Descartes, Paris-V,

15 Rue de l’Ecole de Médecine, 75006 Paris, France.

TEL (33) 1 42 34 69 31 FAX (33) 1 43 25 63 44;

82 Abstract

Interaction between tumor cells and their microenvironment have a crucial role in the development, progression and drug resistance. Our research objective was to confirm the role in drugs resistance and tumor cell growth of hospicells, which are stromal cell from cancer microenvironment. We demonstrated that soluble factors secreted by hospicells activate several genes and favour JAK-STAT signaling pathway in ovarian cancer cell lines. Hospicells express all insulin like growth factor (IGF) family as detected by gene array, RT-PCR, protein array and immunocytochemistry. However, whether IGF-I is secreted by hospicells was not clear. While focussing attention on the microenvironment, we considered the role of IGF-I in proliferation and survival of ovarian cancer cells. Indeed IGF-I is a major actor in modulating different stages of cancer development. We studied the effect of exogenously added IGF-I on the regulation of ATP binding cassette ABC gens (MDR1, MRP1, 2, 3, 5 and BCRP) in the ovarian cancer cell line (OVCAR3) and confirmed the results obtained making use of the IGF-IR antagonist, picropodophyllin. IGF-1 regulates the expression of ABC genes in OVCAR3 cells via PI3-Kinase, MEK and JAK2-STAT3 signaling pathways. OVCAR3 cell line when co-cultivated with hospicells, showed marked degree of drugs resistance development. This drugs resistance could be amplified if IGF-I was exogenously added. Addition of IGF-IR inhibitor however reduced the degree of resistance in these exposed cells. At the same time cells treated with anti cancer drugs but then exposed to IGF-I showed an increase in drugs resistance and thereby increase in cell survival. This observation indicates that drug resistance of OVCAR3 cells increases when there is synergy between OVCAR3 cells and hospicells and it is amplified when IGF-I was exogenously added. In conclusion, inhibition of IGF-IR and targeting of JAK-Stat signaling pathway can be a target for ovarian cancer therapy.

83

Introduction

The tumor microenvironment has been largely studied as a dynamic system orchestrated by several growth factor as Insulin like Growth Factor-I (IGF-I) (Uchiyama et al, 1993; Chauhan et al, 1996; Dankbar et al, 2000; Gupta et al, 2001). IGF-I functions by autocrine and/or paracrine effect, in physiological and pathological processes, but is also involved in development and progression of several cancers as ovarian cancer (Druckmann, and Rohr, 2002). High levels of free IGF-1 and IGF-1 activity are associated with elevated risk of progression in ovarian cancer (Brokaw et al., 2007). In Plasma, IGF-1 binds mainly to the IGF binding protein, IGFBP-3 (Holly and Perks, 2006). Ovarian cancer is the sixth most frequent tumors in women and the first cause of death from the gynaecological cancers in the world (Jemal et al., 2008). The efficiency of treatments is often hindered due to emergence of multi resistance (MDR). The microenvironment surrounding the tumor plays a significant role in MDR development Rafii et al., 2008 and also in the progression and metastasis. Peritoneal homeostasis is influenced by growth factors and stromal cells. The peritoneal stromal cells present in ascetic fluids of ovarian cancer have been shown to stimulate cell growth (Sako et al., 2003; Wilson, 1989). A distinctive population of stromal cells forming the tumor microenvironment of floating cell aggregates from ascitic fluid was identified and isolated

for the first time in our laboratory and named “Hospicells” (Rafii et al., 2008, Pasquet et al., 2008,

Lis et al, 2010, Martinet et al, 2010, Castells et al, 2012). The hospicells have been shown confer drug resistance to cancer cells during chemotherapy by trogocytosis (cell-cell contact). At present we provide data demonstrating that IGF-1 up regulates MDR1, MRP1, MRP2 and BCRP from of ATP binding cassette gene family via PI3-Kinase, MEK and JAK- STAT3 pathway. Hospicells and ovarian cancer cells are mutually synergic in the presence of each other for the development of multi drugs resistance.

84

Material and Methods

1. Cell Culture cell lines

The cell line of human ovarian adenocarcinoma (OVCAR3) was from the American Type Culture Collection (ATCC). OVCAR3-eGFP cells, genetically modified by lentiviral vector encoding eGFP (Genethon, Evry), were also used in this study. These two cell lines were maintained in culture using RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% L-glutamine and 50 Units /ml antibiotics (penicillin and streptomycin) and incubated at 37 °C in an atmosphere humidified with 5% CO2.

Hospicells isolated from ascites

The Hospicells were isolated from ascites of patients with ovarian cancer at stage III. These cells were obtained according to the protocol described in the article by Rafii and al. (2008). These hospicells were immortalized with T antigen of the SV-40 virus (P-BABE largeT SV40, Addgene, USA) and were named M16 cells.The M16 cells were grown in RPMI-1640 medium at 37°C in 5% CO2.

Preparation of supernatant from hospicells (M16 cells)

M16 cells (106 cells / ml of culture medium) were incubated for 24 hours at 37° C in RPMI without FBS. After incubation, the supernatant was collected and stored at -80° C. The supernatant was studied for its effect on resistance of OVCAR3 cells against chemotherapy drugs in the presence of IGF-I or in the presence of inhibitor of IGF-IR, called cyclolignan picropodophyllin (PPP).

2. Treatment of OVCAR3 cells with IGF-I and inhibitor of signaling pathways

The adherent OVCAR3 cells were incubated with IGF-I (200 ng/ml) and the following inhibitors from Calbiochem (Paris, France): 1μM of picropodophyllin (an inhibitor of IGF-IR);

5μM of wortmannin (PIγ kinase inhibitor), 5μM of rottlerin (PKC inhibitor); 5μM of PD98059

85

.

Total RNA was extracted with the Nucleospin RNA II kit (Macherey-Nagel EURL, Hoerdt, France). Reverse transcription was performed using M-MLV reverse transcriptase and oligo (dT) primers (Gibco, Paisley, UK). The polymerase chain reaction (PCR) was performed by Taq DNA polymerase (Gibco-BRL, Paisley, UK). Specific primers for IGF-I, (sense : 5’-AAA TCA GCA G TC TTC CAA C-γ’, antisense : 5’-CTT CTG GGT CTT GGG CAT GT-γ’; IGF-II (sense : 5’- AGT CGA TGC TGG CTT CTC A-γ’, antisense : 5’-GTG GGC GGG GTCT TGG GTG GGT AG-γ’); IGF-IR (sense : 5’-GAC ATC CGC AAC GAC TAT CAG-γ’, antisense : 5’-GTA GTT ATT GGA CAC CGC ATC-γ’); IGF-IIR (sense : 5’-TAC AAC TTC CGG TGG TAC ACC A-γ’,

antisense : 5’-CAT GGC ATA CCA GTT TCC TCC A-γ’); MDR1(5’-GTT ATA GGA AGT TTG

AGT TT-γ’ and antisense : 5’-AAA AAC TAT CCC ATA ATA AC-γ’); MRP (5'-AAT GCG CCA AGA CTA GGA AG -3' and antisense 5'-ACG GGA GGA TGT TGA ACA AG-3'); MRP2

(sense : 5’- CTG GTT GAT GAA GGC TCT GA-γ',antisense : 5’-CTG CCA TAA TGT CCA

GGT TC-3'); MRP3 (sense : 5'-GCA GGT GAC ATT TGC TCT GA-3' antisense : 5'- CCC TCT GAG CAC TGG AAG TC-3'); MRP5 (sense : 5'-GGA TAA CTT CTC AGT GGG- 3', antisense : 5'-GGA ATG GCA ATG CTC TAA AG-3') ; BCRP (sense : 5'-TTA GGA TTG AAG CCA AAG G-3', antisense : 5'-TAG GCA ATT GTG AGG AAA ATA-3') and β2-microglubulin (sense : 5'- CCA GCA GAG AAT GGA AAG TC-3', antisense : 5'-GAT GCT GCT TAC ATG TCT CG-3'). The PCR products, along with a 100-bp DNA ladder, were analysed by electrophoresis on agarose gels containing ethidium bromide.

4. IGF Protein expression by OVCAR3 cells

The presence of proteins belonging to IGF family in OVCAR3 cells was revealed by immunocytochemistry. OVACR3 were seeded at 20,000 cells / well in glass bottom chamber slides (Lab-Tek, Nunc, Naperville, USA). The cells were then permeabilized and incubated for 2 h at room temperature with either specific primary antibodies (dilution 1/200) anti-IGF-I, -II, -IR or - IIR (R & D Systems, Minneapolis, USA). After several washes, cells were incubated successively with biotinylated secondary antibody and streptavidine coupled to fluorescein isothiocyanate (dilution 1/500), for 45 min. Isotypic controls were performed concurrently and nuclei were DAPI- labeled. The cells were then visualized by fluorescence microscopy.

86 5. Analysis by Gene Array of genes expression in OVCAR3 cells incubated in the

hospicells supernatant

Expressions of several genes were analysed by gene microarray (Microarray PIQOR; Miltenyi Biotec. GmbH, Bergisch Galdbach, Germany) in OVCAR3 cells incubated in the hospicells supernatant for 8 h at 37°C. 5x106 of OVCAR3 cells were lysed with Trizol® reagent (Invitrogen, Carlsbad, USA) and sent to Miltenyi Biotec GmbH in dry ice. The gene array experiment was performed according to the protocol of Ducros et al., 2007. Expressions of IGF-I, IGF-II, IGF-IR, IGF-IIR and IGFBP 1, 2, 3, 4, 6, 10 genes were also studied by gene array.

6. Quantitative Analysis by protein Array of IGF family proteins secreted by M16 cells We analysed the supernatant of M16 cells to measure the amount of IGF family proteins (IGF-I, IGF-II, IGF-IR and IGF-IIR) using protein array technology (Human Cytokine Antibody Array, Ray Biotech, Norcross, GA). This technique is based on the principle of ELISA. Membranes on which 174 anti-cytokines were fixed with appropriate controls were used. The M16 cell supernatant, prepared above, contained proteins that were linked with their specific antibodies attached to the membrane. The membrane was saturated for 2 h with BSA at room temperature to block nonspecific sites. Then, the M16 cell supernatant was incubated overnight at 4° C with antibodies specific for IGF-I, IGF-II, IGF-IR. After several washes, the membranes were incubated with a mixture of biotinylated secondry antibodies overnight at 4° C. Streptavidin coupled to HRP was added to the membranes, while being incubated for 2 h at room temperature.

The presence of a proteins coupled to the antibody was revealed using the ECL reagent membrane (Enhanced Chemiluminescence, GE Healthcare Europe, Saclay, France). The membrane was dried and then exposed on a photographic film (Amersham Biosciences, Montigny-le-Bretonneux, France) before being developed (Kodak X-OMAT Processor 1000). The ImageJ software (U.S. National Institutes of Health, Bethesda, USA) was used to quantify the intensity of the spot and compared to control experiments.

7. Studies of drug resistance of OVCAR3-eGFP cells in the presence of Hospicells

The hospicells were seeded first at 60% confluency in a 96-well flat-bottomed cells-culture plate in the presence of complete medium containing 10% FBS. After 18 hours incubation, the OVCAR3-

87 eGFP cells (10 000 cells / well) were added and co-cultured for 24 h with hospicells in the presence of IGF-I (200 µg/ml) or IGF-IR inhibitors : (PPP 1μM) or (siRNA, 10nM- HiPerFect, Qiagen, Courtaboeuf, France) before addition of ββ.β µM carboplatin or 1.4 μM paclitaxol (Smith et al., 2005). The effect of these cytotoxic agents was evaluated quantitatively using a GFP fluorescence reader (Wallac, Perkin&Elmer, Waltham, USA). The same experiment was also performed on OVCAR3 cells incubated in the presence of supernatants of hospicells as described above. The result is representative of three independent experiments.

7.

The results are presented as mean ± SE and data was analysed using student test P value <0.05 was considered significant.

Results

1. Effect of conditioned medium from hospicells on OVCAR3.

The aim of this experiment was to study the effect of conditional medium from hospicells on the regulation of selected gens in OVCAR3 cells in vitro by gene array. The major genes up or down regulated after interaction of soluble factors secreted by hospicells on ovarian cancer cells are presented in table 1. Several genes including those for cytokine and cytokine receptors such as PDGFB, TNFSF5, VEGF-2, GPR17, gpl 130 (IL6 ST), IGFBP10 and ABCc10 (MRP-7), are up- regulated. Curiously, the amount of several genes encoding for transcription factors such as RASA4, STAT2, STAT6, IRF4, and MARK3 were found increased. In contrast, the expression of IL2, IL13, PAR3, ABCB3 (MDR-3) were decreased. In a parallel study using code genes for all up regulated mRNAs in adatabase for annotation, visualization and integrated discovery (DAVID) v6.7 analysis, JAK-STAT signaling pathway was suggested.

88 2. Expression of IGF related proteins by hospicells .

In the previous experiments, we observed that the conditional medium of hospicells regulate of MRP-7 and MDR-3 mRNA expression after 8h incubation with OVCAR3 cell line. Because of the importance of IGF signaling pathway in cancer cell proliferation and metastasis, we concentrated our works on IGF family. Gene array, protein array, RT-PCR and immunocytochemistry methods used for detection of IGF related family in Hospicells. All results are resumed in table 2. IGF-I, IGF-II, IGF-IR, IGF-IIR and IGFBP 1, 2, 3, 4, 6 and 10 were detected in gene array. The expressions of all proteins were confirmed by protein array. RT-PCR was used to detect the presence of mRNA of IGF family members (IGF-I, IGF-II, IGF-IR and IGF-IIR) in hospicells isolated from ascites of patients with ovarian cancer and β-microglubuline was implied as a control. Figure 1-A shows that Hospicells (M16 cells) express IGF-I, IGF-II genes as well as their receptors IGF-IR, IGF-IIR. The PCR results were confirmed by immunocytochemical analysis using specific antibodies for IGFs and their receptors (Figure 1-B). These results showed that several member of IGF family were expressed by hospicells.

3. IGFs and their receptors expressed in the ovarian cancer cell line and implicated in cell proliferation

The presence of IGF-I, IGF- II and their receptors IGF-IR and IGF-IIR proteins in ovarian cancer cell line OVCAR3 has been determined by immunofluorescence analysis using specific antibodies. IGF-I, IGF-II and IGF-IR are expressed at significant levels in OVCAR3 cell lines (Figure 2-A). In contrast, the immunofluorescence of the IGF-IIR was low, indicating that these cells express the receptor rather weakly. In parallel, we studied, using the MTT technique, the effect of the growth factor IGF-I on OVCAR3 cell proliferation. This experiment was performed in serum-free medium to avoid skewing the results of the study. We also investigated whether inhibition of IGF-IR with picropodophyllin (PPP) decreased OVCAR3 cell proliferation. The result in Figure 2B shows that OVCAR3 cells are sensitive to the effect of IGF-I and their proliferation increases at time 48h. We also found that inhibition of IGF-IR, indispensable for transmitting the effects of IGF-I on cells, leading to a strong reduction in the growth and proliferation of OVCAR3 cells.

89 4. Expression of ABC genes in OVCAR3 cells

ABC transmembrane proteins play an important role in drug resistance of cancer cells because they expel from the cells the anticancer drugs. Indeed, over-expression of ABCB1 (MDR1 gene and its P-gp protein) has been directly involved in the phenomenon of multidrug resistance in vitro. Ovarian cancer cell line OVCAR3 is characterized by an average degree of aggressiveness. We sought to determine the expression of ABC genes by OVCAR3 cells with RT-PCR using primers specific for MDR1, MRP1, MRP2, MRP3, MRP5 and BCRP (see Material and Methods). The amount of mRNA for β2-microglobuline was also analyzed to normalise the results. The results in Figure 3 show that OVCAR3 cells do not express MDR1 and MRP2 genes at a significant level. The expression of MRP1 and MRP3 is above basal levels wherease expression of MRP5 and BCRP genes are strong. The results shown also in Figure 3 present the expression of ABC genes when the OVCAR3 cells treated with IGF-1. In the presence of the inhibitor PPP, the expression of the gene MDR1 disappears. We obtained a similar result for the expression of MRP2. We also found that IGF-I increases the expression of genes MRP3 and MRP1. This expression is greatly reduced in the presence of the inhibitor of IGF-IR when compared to control cells. In contrast, there was no significant difference in expression of MRP5 and BCRP in the presence of IGF-I or PPP compared to control cells. This suggests that MRP-1 and MRP-3 gene promotors are activated by the IGF-I pathway. The intensity of bands in each experiment was evaluated using ImageJ. The results were normalised with the expression of the mRNA of β-microglobuline.

5. IGF-1 regulate the expression of ABC genes in OVCAR3 cells by PI3-Kinase, MEK and JAK2-STAT3 pathways

To elucidate the mechanism of drug resistance facilitated by the expression of ABC genes in OVCAR3 cells in the presence of IGF-1 or its absence (negative control), we tested the effects of five inhibitors of signaling pathways: rottlerin (PKC inhibitor), wortmannin (PI3 kinase inhibitor), PD98059 (MEK inhibitor), Jak2 inhibitor and STAT3 inhibitor. ABC gene expressions were induced at levels different by IGF-I (Figure 4). Indeed, the inhibitor PD98059 decreased the expression of three ABC genes MDR1, MRP1 and MRP2. The inhibitor Vortmannin in the presence of IGF-I inhibits completely the expression of BCRP and MRP2 in OVCAR3 cells and. Jak 2 inhibitor also reduces the expression of MDR1 and MRP2. Our attention has been retained by the effect of the STAT3 inhibitor on expression of MDR1 and MRP1. The expression of these

90 genes was totally inhibited by the addition of STAT3 inhibitor. This is important since MDR1 and MRP1 are known for their role in the resistance of ovarian cancer against chemotherapy, including carboplatin and taxoplatine. In contrast, no difference in MRP3 and MRP5 genes expression was observed in the presence of various inhibitors as compared to OVCAR3 control cells incubated only with IGF-I.

6. The effect of IGF-I on drug resistance of OVCAR3 cells with conditioned medium from hospicells

Inhibition of IGF-IR (or signaling pathways activated by this receptor) can alter ABC gene expression, mainly MDR1. The OVCAR3 cells were incubated in a serum free medium (control cells) or with supernatants of hospicells in the presence of IGF-I or PPP. The incubation of